Phosphorylation of the 20,000 dalton subunit (light chain) of myosin is being studied in smooth muscle strips dissected from pit carotid arteries. Phosphorylation of the light chain is being measured by densitometry of two-dimentional isoelectric focusing-SDS electrophoresis slab gels. This procedure separates phosphorylated from non-phosphorylated light chains in homogenates of whole muscles. The homogenates are prepared from muscles which are quickly frozed in various contractile states. The method avoids the use of radioisotopes and directly gives the amount of phosphorylated and non-phosphyorylated light chains in small samples of tissue, typically 5-10mg wet weight. The mechanical responses of the smooth muscle tissues are quantified by measurements of the length-tension and force-velocity relationships. Stiffness measurements will also be made. The mechanical responses are measured at various times after stimulation with various agonists and compared to the extent of phosphorylation of the light chains. These studies are elucidating the role of myosin light chain phosphorylation in the regulation of contraction of intact smooth muscle tissue. The results to date show that phosphorylation occurs on stimulation, but suggest that other mechanisms are involved in the maintenance of isometric force.